Fluorescent dyes have been widely used in biological research and clinical diagnosis in which a high sensitive fluorescence detection is desirable. Compared to radioactive detection, fluorescent dyes are less expensive and less toxic. In particular, a diversity of fluorescence dyes with different emission wavelengths has been successfully used for multiplex detection in parallel, such as immunoassay, PCR reaction, SNP, and DNA sequencing. The fluorescent dyes with fluorescence emission maxium in the near infrared region have also been used for in vivo imaging. In addition, the fluorescent dyes have important applications in high-throughput screening.
Despite the diverse applications of fluorescent dyes, the conventional dyes have some limitations in biological applications. First, the conventional dyes are easily prone to self-quenching by dimer formation, a phenomenon known to diminish the effective brightness of the dyes. When the conventional dyes are conjugated with biomolecules such as protein, the fluorescence intensity of the labeled target is not directly to the number of attached dye molecules, but rather less than the predicted intensity due to self-quenching effect amongst the multiple dyes attached to the target. Second, the typical emission band half-width of the conventional dyes is about 40-80 nm. When using these conventional dyes for multiplex application, it is difficult to find a set of dyes whose emission spectra are spectrally resolved. Third, the low fluorescence quantum yield of conventional dyes decreases the detection sensitivity. All those limit the applications of conventional dyes.
Adding sulfonate groups to a dye has been shown to decrease the inherent tendency of the dye to form dimmers and aggregate, presumably due to the increased polar sulfonic acid moiety. See, e.g., U.S. Pat. Nos. 5,268,486, 6,977,305, and 6,130,101, and Panchuk-Voloshina, et al. J. Histochem. Cytochem. 47(9), 1179 (1999). Sulfonation increases the water solubility and the fluorescence brightness of carbocyanine dyes, but the photo-stability of carbocyanine dyes remains to be improved.
Rhodamine dyes with narrow fluorescence emission spectra can be obtained by the addition of chlorides to the phenyl group. For example, the emission spectra of dR110 is 10-15% narrower and has emission maxima at 10 nm longer wavelength than the unsubstituted R110. For more examples, see U.S. Pat. Nos. 5,847,162, 6,017,712, 6,025,505, 6,080,852, and 6,713,622, and L. G. Lee, et al. Nucleic Acids Res. 25(14), 2816 (1997). When using these 4,7-dichlororhodamine dyes to conjugate with proteins, these dyes are also prone to self-quenching by dimer formation.
The present invention describes 4,7-dichlororhodamine dyes that are substituted by at least one sulfonate moiety. The sulfonated 4,7-dichlororhodamine dyes of the invention possess considerable advantages over their non-sulfonated analogs, such as greater water solubility, and brighter fluorescence intensity. In particular, the sulfonated 4,7-dichlororhodamine dyes of the invention possess better photo-stability than those of other dyes having comparable spectra, such as cyanine dyes. In addition, the sulfonated 4,7-dichlororhodamine dyes of the invention exhibit resistance to quenching upon protein conjugation. Besides, the sulfonated 4,7-dichlororhodamine dyes of the invention are much easier to separate the 5-isomer and 6-isomer than the unsulfonated 4,7-dichlororhodamine dyes, due to good water solubility.